title: Thyrotropin-releasing hormone stimulation of prolactin release from clonal rat pituitary cells: evidence for action independent of extracellular calcium.
abstract: Thyrotropin-releasing hormone (TRH) stimulates prolactin release and (45)Ca(2+) efflux from GH(3) cells, a clonal strain of rat pituitary cells. Elevation of extracellular K(+) also induces prolactin release and increases (45)Ca(2+) efflux from these cells. In this report, we distinguish between TRH and high K(+) as secretagogues and show that TRH-induced release of prolactin and (45)Ca(2+) is independent of the extracellular Ca(2+) concentration, but the effect of high K(+) on prolactin release and (45)Ca(2+) efflux is dependent on the concentration of Ca(2+) in the medium. The increment in (45)Ca(2+) efflux induced by 50 mM K(+) during perifusion was reduced in a concentration-dependent manner by lowering extracellular Ca(2+) from 1,500 to 0.02 muM (by adding EGTA), whereas 1 muM TRH enhanced (45)Ca(2+) efflux similarly over the entire range of extracellular Ca(2+) concentrations. Although 50 mM K(+) caused release of 150 ng prolactin from 40 x 10(6) GH(3) cells exposed to 1,500 muM Ca(2+) (control), reduction of extracellular Ca(2+) to 2.8 muM decreased prolactin release caused by high K(+) to <3% of controls and no prolactin release was detected after exposure to 50 mM K(+) in medium with 0.02 muM free Ca(2+). In contrast, TRH caused release of 64 ng of prolactin from 40 x 10(6) GH(3) cells exposed to medium with 1,500 muM Ca(2+), and release caused by TRH was still 50 and 35% of control in medium with 2.8 and 0.02 muM Ca(2+), respectively. Furthermore, TRH transiently increased by 10-fold the fractional efflux of (45)Ca(2+) from GH(3) cells in static incubations with 1,500 or 3.5 muM Ca(2+), hereby confirming that the enhanced (45)Ca(2+) efflux caused by TRH in both low and high Ca(2+) medium was not an artifact of the perifusion system.Data obtained with chlortetracycline (CTC), a probe of membrane-bound Ca(2+), were concordant with those obtained by measuring (45)Ca(2+) efflux. Cellular fluorescence of CTC varied with the extracellular Ca(2+) concentration and the duration of incubation. TRH decreased the fluorescence of cell-associated CTC in a manner strongly suggesting stimulus-induced mobilization of Ca(2+), and this effect was still demonstrable in GH(3) cells incubated in 50 mM K(+). These data suggest that TRH acts to mobilize sequestered cell-associated Ca(2+) reflected as a (45)Ca(2+) efflux which is independent of the extracellular Ca(2+) concentration. Mobilization of sequestered Ca(2+) into the cytoplasm may elevate free intracellular Ca(2+) and serve to couple stimulation by TRH to secretion of prolactin.
mesh_terms: D000818:Animals; D002118:Calcium / Q000032:analysis / Q000378:metabolism* / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002751:Chlortetracycline; D010902:Pituitary Gland / Q000378:metabolism*; D011188:Potassium / Q000494:pharmacology; D011388:Prolactin / Q000378:metabolism*; D051381:Rats; D013050:Spectrometry, Fluorescence; D013973:Thyrotropin-Releasing Hormone / Q000494:pharmacology*
chemical_list: D013973:Thyrotropin-Releasing Hormone; D011388:Prolactin; D011188:Potassium; D002118:Calcium; D002751:Chlortetracycline